This project involves the study of several intracellular lipid-binding proteins from the small intestine. These proteins have distinct specificities, stoichiometries and affinities for fatty acids, steroids, retinoids and eicosanoids, but the basis for these differences is unclear. The long-term goal of this research is to define the rules that govern molecular recognition in this protein family. The specific aims are (1) to characterize a panel of mutants designed to perturb the dynamic portal of rat and human intestinal fatty acid-binding protein (I-FABP); (2) to engineer variants of rat and human I-FABP with increased affinity for fatty acids, and to develop prototype high- affinity ligands for wild-type I-FABP; (3) to engineer a second- generation helix-less variant of I-FABP and use it as a model system for investigating the determinants of lipid-binding specificity; (4) to characterize the interactions between a physiologically relevant bile salts and ileal lipid-binding protein (I-LBP); and (5) to determine the 3D structures and backbone mobilities of human I-LBP with and without bound bile salts. The experimental approach employs triple-resonance NMR, fluorescence spectroscopy and isothermal calorimetry to characterize these proteins and their complexes with lipids. Definition of the roles for lipid-protein recognition may lead to the design of specific high affinity inhibitors with therapeutic potential in type II diabetes and hypercholesterolemia.